Aromatic acids

ABSTRACT

The invention relates to novel aromatic acids, especially to compounds of formula ##STR1## wherein R 1  is lower alkyl, R 2  is lower alkyl, R 3  is hydrogen, carboxy or sulfo, R 4  is carboxy or sulfo, G is an unsubstituted or substituted 1,4-phenylene group or an unsubstituted or substituted 1,4-naphthylene group, and wherein either R 5  and R 6  together are an additional bond and L is an oxygen or sulfur atom or wherein R 5  is hydrogen, R 6  is halomethyl and L is an oxygen atom, and salts thereof, to the use of compounds I and their salts, to a process for the preparation of compounds I and their salts, to starting materials used in that preparation process, and salts thereof, to a process for the preparation of those starting materials and their salts, to a device in which the compounds I and their salts are used, and to a process in which that device is used. The compounds of formula I can be used as adjuncts in the investigation of proteins and can be prepared in a manner known per se.

This is a DIVISIONAL of Ser. No. 255,298, filed Jun. 7, 1994, now U.S.Pat. No. 5,382,659, which is a CONTINUATION of Ser. No. 056,725, filedMay 3, 1993, now abandoned, which is a DIVISIONAL of Ser. No. 758,249,filed Sep. 11, 1991 now U.S. Pat. No. 5,229,503, which is a CONTINUATIONof Ser. No. 348,315, filed May 5, 1989 now abandoned.

The invention relates to novel aromatic acids, especially compounds offormula ##STR2## wherein R₁ is lower alkyl, R₂ is lower alkyl, R₃ ishydrogen, carboxy or sulfo, R₄ is carboxy or sulfo, G is anunsubstituted or substituted 1,4-phenylene group or an unsubstituted orsubstituted 1,4-naphthylene group, and wherein either R₅ and R₆ togetherare an additional bond and L is an oxygen or sulfur atom or wherein R₅is hydrogen, R₆ is halomethyl and L is an oxygen atom, and saltsthereof, to the use of compounds I and their salts, to a process for thepreparation of compounds I and their salts, to starting materials usedin that preparation process, and salts thereof, to a process for thepreparation of those starting materials and their salts, to a device inwhich the compounds I and their salts are used, and to a process inwhich that device is used.

Suitable unsubstituted or substituted 1,4-phenylene groups are, forexample, unsubstituted or carboxy- and/or sulfo-substituted1,4-phenylene groups, the substituted 1,4-phenylene groups having from 1up to and including 4, especially 1 or 2, of the mentioned substituents.If the substituted 1,4-phenylene groups contain more than onesubstituent, then some or all of those substituents may be identical.Examples that may be mentioned are the 2-sulfo-, 2,3-, 2,5- and3,5-disulfo-, 2,3,5-trisulfo- and 2,3,5,6-tetrasulfo-1,4-phenylenegroup, the 2-carboxy-, 2,3-, 2,5- and 3,5-dicarboxy-, 2,3,5-tricarboxy-and 2,3,5,6-tetracarboxy-1,4-phenylene group, the 2-carboxy-3-sulfo-,2-carboxy-5-sulfo-, 3-carboxy-5-sulfo-, 2,3-dicarboxy-5-sulfo-,3,5-dicarboxy-2-sulfo-, 5-carboxy-2,3-disulfo- and2-carboxy-3,5-disulfo-1,4-phenylene group and especially the1,4-phenylene group.

Suitable unsubstituted or substituted 1,4-naphthylene groups are, forexample, unsubstituted or carboxy- and/or sulfo-substituted1,4-naphthylene groups, the substituted 1,4-naphthylene groupscontaining from 1 up to and including 6, especially from 1 up to andincluding 3, of the mentioned substituents. If the substituted1,4-naphthylene groups contain more than one substituent, then some orall of those substituents may be identical. Examples that may bementioned are the 2-, 5- and 6-sulfo-, 2,3-, 5,6-, 6,7- and2,6-disulfo-, 2,3,5- and 2,3,6-trisulfo- and 2,3,5,7- and2,3,6,7-tetrasulfo-1,4-naphthylene group, the 2-, 5- and 6-carboxy-,2,3-, 5,6-, 6,7- and 2,6-dicarboxy-, 2,3,5- and 2,3,6-tricarboxy- and2,3,5,7- and 2,3,6,7-tetracarboxy-1,4-naphthylene group, the2-carboxy-3-sulfo-, 2-carboxy-5-sulfo-, 3-carboxy-6-sulfo-,5-carboxy-7-sulfo-, 2,3-dicarboxy-5-sulfo-, 3,5-dicarboxy-2-sulfo-,6,7-dicarboxy-2-sulfo-, 3-carboxy-6,7-disulfo-,5-carboxy-2,3-disulfo-and 2-carboxy-3,5-disulfo-1,4-naphthylene groupand especially the 1,4-naphthylene group.

The invention relates, for example, to compounds I wherein R₁ is loweralkyl, R₂ is lower alkyl, R₃ is hydrogen, carboxy or sulfo, R₄ iscarboxy or sulfo, G is an unsubstituted or substituted 1,4-phenylenegroup or an unsubstituted or substituted 1,4-naphthylene group, R₅ andR₆ together are an additional bond and L is an oxygen or sulfur atom,and salts thereof.

Some of the compounds I can be in the form of stereoisomers. Forexample, if the compounds I contain at least one chiral carbon atom (Catom) (for example a C atom of a corresponding radical R₁), they can be,for example, in the form of pure enantiomers or mixtures of enantiomers,such as racemates, and if there is at least one further chiral centrepresent (for example a C atom of a corresponding radical R₂), they mayalso be in the form of diastereoisomers, mixtures of diastereoisomers ormixtures of racemates.

Salts of compounds I are especially salts with bases, preferablypharmaceutically acceptable salts with bases, for example alkali metalsalts or alkaline earth metal salts, for example sodium, potassium ormagnesium salts, transition metal salts, such as zinc or copper salts,or salts with ammonia or organic amines, such as cyclic amines, such asmono-, di- or tri-lower alkylamines, such as hydroxy-lower alkylamines,for example mono-, di- or tri-hydroxy-lower alkylamines, hydroxy-loweralkyl-lower alkylamines or polyhydroxy-lower alkylamines. Cyclic aminesare, for example, morpholine, thiomorpholine, piperidine or pyrrolidine.Suitable mono-lower alkylamines are, for example, ethylamine ortert.-butylamine; suitable di-lower alkylamines are, for example,diethylamine or diisopropyl-amine; and suitable tri-lower alkylaminesare, for example, trimethylamine or triethylamine. Suitablehydroxy-lower alkylamines are, for example, mono-, di- ortri-ethanolamine, and hydroxy-lower alkyl-lower alkylamines are, forexample, N,N-dimethylamino- or N,N-diethyl-amino-ethanol, whilst asuitable polyhydroxy-lower alkylamine is, for example, glucosamine. Thecompounds I can also form acid addition salts, preferablypharmaceutically acceptable acid addition salts, for example with stronginorganic acids, such as mineral acids, for example sulfuric acid, aphosphoric acid or a hydrohalic acid, with strong organic carboxylicacids, such as lower alkanecarboxylic acids, for example acetic acid,saturated or unsaturated dicarboxylic acids, for example malonic, maleicor fumaric acid, or hydroxycarboxylic acids, for example tartaric orcitric acid, or with sulfonic acids, such as lower alkanesulfonic acidsor unsubstituted or substituted benzenesulfonic acids, for examplemethane- or p-toluene-sulfonic acid. The compounds I can also form innersalts.

Also included are salts of compounds I that are less suitable forpharmaceutical uses. These may be used, for example, for the isolationor purification of free compounds I according to the invention and theirpharmaceutically acceptable salts.

Hereinbefore and hereinafter, unless defined otherwise, radicals orcompounds designated "lower" are to be understood as being especiallythose radicals or compounds containing up to and including 7, especiallyup to and including 4, carbon atoms.

Lower alkyl is, for example, C₁ -C₄ alkyl, such as methyl, ethyl,n-propyl, isopropyl, n-butyl, isobutyl, sec.-butyl or tert.-butyl, andalso includes C₅ -C₇ alkyl radicals, that is to say pentyl, hexyl andheptyl radicals.

Halomethyl is fluoro-, chloro- or bromo-methyl, but especiallyiodomethyl.

The compounds I and their salts have valuable properties. For example,they can be used as adjuncts in the investigation of proteins, forexample as reagents for the chemical modification of proteins.Hereinbefore and hereinafter the term "protein" is to be understood asincluding both peptides having a relative molecular weight of 10 000 ormore atomic mass units, which are generally termed proteins, andpeptides having a relative molecular weight of less than 10 000 atomicmass units, which are generally termed polypeptides, a lower limit offrom approximately 1000 to approximately 2000 atomic mass units applyingto the relative molecular weight of the polypeptides. Examples takenfrom the numerous applications in which the chemical modification ofproteins can play a part are the structural analysis and the colorationof proteins.

It is known that the modification of a single amino acid building blockor of a small number thereof can substantially alter the spatialstructure of a protein and therefore also its function, for example itsbiological activity. This opens up the possibility of using the specificchemical modification of amino acid building blocks as a widelyapplicable method of determining the contribution made by these buildingblocks to the spatial structure of a protein. The problem arises here ofrelating any alteration in the spatial protein structure occurring as aresult of a certain chemical modification of amino acid building blocksto the nature and extent of that chemical modification. For this purposethe structure of a corresponding chemically modified protein must bedetermined and compared with the structure present before the chemicalmodification was carried out. One of the aims of this kind of structuralanalysis is to determine in what way the protein primary structure haschanged during the chemical modification, that is to say which aminoacid building blocks have been chemically modified. In primary structureanalyses of this kind, the procedure is often as follows: a chemicallymodified protein M_(a), obtained after chemical modification of acorresponding unmodified protein N_(a), and the unmodified protein N_(a)are, optionally after having carried out a denaturation step which maybe necessary, each treated with the same protease, the two peptidemixtures M_(a) ¹ and N_(a) ¹ so obtained are each subjected tohigh-performance liquid chromatography (HPLC) and the peak patterns inthe two resulting chromatograms M_(a) ² and N_(a) ² are compared withone another. The detection systems used for recording thesechromatograms are usually detection systems which evaluate the lightabsorption behaviour of the peptides. The peak pattern in chromatogramM_(a) ² generally differs from the peak pattern in chromatogram N_(a) ²at those places at which peptides M_(a) ³, which contain at least onechemically modified amino acid building block, are detected, since thepeptides M_(a) ³ generally exhibit a light absorption behaviourdifferent from that of the corresponding unmodified peptides N_(a) ³.The peptides M_(a) ³ are separated off and subjected to further primarystructure analysis, for example to amino acid sequence analysis.Ideally, each chemically modified amino acid building block can beidentified and characterised in this manner.

In many cases, however, the usefulness of the above-described procedureleaves something to be desired. For example, when the protein to beinvestigated has a relatively high relative molecular weight, there arenormally so many different peptides present in the corresponding peptidemixture after the protease treatment that HPLC is unable to effectsufficient separation of the peptide mixture. Furthermore, the chemicalmodification itself is often too complex; it often proceeds in anonspecific manner and therefore affects a large number of structurallydifferent amino acid building blocks or even all the amino acid buildingblocks in the protein under investigation; advantageously, however, itshould be possible to carry out the chemical modification asspecifically as possible, that is to say specifically directed only at aclass of amino acid building blocks that is defined as accurately aspossible and advantageously that is narrowly defined in terms of natureand number.

It is therefore desirable and of great practical interest to optimisethe procedure described above by overcoming the disadvantages indicated.The compounds I disclosed within the framework of the present inventionand their salts make such an optimisation possible. This optimisation isexplained in detail below with reference to compounds I in which R₁, R₂,R₃, R₄ and G are as defined above, R₅ and R₆ together are an additionalbond and L is an oxygen or sulfur atom, and salts thereof. Thesecompounds are designated compounds IA below.

In the chemical modification of a protein, that is to say in thereaction with that protein, the compounds IA and their salts react inthe first instance virtually exclusively with the amino groups in theε-position of lysine building blocks, with high reactivity beingobserved. Only in isolated cases is there additionally a reaction withthe amino group of the N-terminal amino acid building block of theprotein in question. The nature and number of the amino acid buildingblocks of a protein that are affected by a chemical modification by acompound IA or a salt thereof are therefore clear and, advantageously,narrowly defined. Thus the number of chemically modified amino acidbuilding blocks is generally not greater than the number of lysinebuilding blocks in the protein under investigation. Only in isolatedcases, when there is an additional N-terminal modification, can it begreater by 1.

Furthermore, the use of a compound IA or a salt thereof for the chemicalmodification of a protein has the great advantage that after theprotease treatment of the resulting chemically modified protein M_(b)there is obtained a peptide mixture M_(b) ¹ in which by means of simplereversed phase HPLC it is possible to obtain separation, usuallycomplete separation, of the peptides M_(b) ³, which contain at least onechemically modified amino acid building block, from the remainingpeptides N_(b) ³.

In addition, the peptides M_(b) ³, which contain at least one chemicallymodified amino acid building block, present in the peptide mixture M_(b)¹ on the one hand and the unmodified peptides N_(b) ³ on the other handcan each be detected at markedly different wavelengths, since thepeptides M_(b) ³ exhibit a light absorption behaviour that is markedlydifferent from that of the peptides N_(b) ³. This considerablyfacilitates the identification of the peptides M_(b) ³. Advantageously,the peptides M_(b) ³, as they have a colour, can be detected atwavelengths of visible light, that is to say at wavelengths betweenapproximately 400 nm and approximately 800 nm for example in acidicsolution at a wavelength of 535 nm or in alkaline solution at awavelength of 465 nm, preferably at one of the wavelengths mentioned inExamples 10 to 15. The colourless peptides N_(b) ³, however, can bedetected at wavelengths of ultraviolet light, for example at awavelength of 200, 220 or 280 nm, and therefore do not interfere withthe detection of the coloured peptides M_(b) ³.

It is also of particular interest that it is not only the peptides M_(b)³ that have a colour but also the corresponding chemically modifiedprotein M_(b) from which the peptides M_(b) ³ are obtained by proteasetreatment. Since the chemical modification of a protein by a compound IAor a salt thereof is therefore always associated with coloration of theprotein, the compounds IA and their salts can also be used as reagentsfor the coloration of proteins. The coloured proteins M_(b) obtainableby reaction of the ε-amino groups of lysine building blocks and, inisolated cases, additionally by reaction of the amino group of theN-terminal amino acid building block of corresponding colourless,unmodified proteins Nb with a compound IA or a salt thereof, can be usedin a variety of ways for analytical and/or diagnostic purposes. Thecomments made above in connection with the separation and detection ofpeptides M_(b) ³ that have a colour also apply in analogous manner tothe separation and detection of the proteins M_(b) that have beencoloured. The peptides M_(b) ³ can also be used for a variety ofanalytical and/or diagnostic purposes.

Since the investigation of proteins generally takes place in aqueous orwater-containing solution, the good water-solubility of the compounds IAand their salts constitutes a further valuable property. Because thechemically modified proteins M_(b) and the peptides M_(b) ³, whichcontain at least one chemically modified amino acid building block, arealso distinguished by good water-solubility, both the reaction ofproteins Nb with a compound IA or with a salt thereof and any otherprocess steps customary in investigations of proteins which might beenvisaged, for example of the kind mentioned below, can advantageouslybe carried out in aqueous solution, optionally with the addition of anorganic solvent. In the course of the reaction with a compound IA orwith a salt thereof, the corresponding amino groups of a protein Nb ofgeneral formula H₂ N--R_(x) (Ia) wherein R_(x) in each case is theresidue of the protein, that is to say the ε-amino groups of lysinebuilding blocks and in isolated cases additionally the amino group ofthe N-terminal amino acid building block, are converted into carbamoylor thiocarbamoyl groups, respectively, so that a chemically modifiedprotein of formula ##STR3## wherein R₁, R₂, R₃, R₄, R_(x) and G are asdefined above and L is an oxygen or sulfur atom, is obtained. The speedof this conversion reaction increases the higher the temperature and thehigher the pH value of the reaction medium. Examples of typical reactionconditions can be found in Examples 9 to 15. The stability of thecarbamoyl or thiocarbamoyl bonds, respectively, in the proteins Ib isadvantageously so great in normal cases that the proteins Ib can bestored for periods of several days without any appreciable decompositiontaking place and that they can also withstand unimpaired the furtherprocess steps customary in investigations of proteins, such aschromatographic separations, for example by means of molecular exclusionchromatography, for example gel chromatography, or HPLC, enzymatictreatments, for example treatment with a protease, for example withtrypsin or chymotrypsin, other standard reactions, for exampledenaturation steps, such as the reduction of disulfide bridges and thesubsequent carboxymethylation of the mercapto groups, or test reactionsfor the analysis of the biological activity. Such process steps areknown or can be carried out analogously to known process steps. Examplesof details of such process steps can also be found in Examples 9 to 15.As regards the stability of the carbamoyl or thiocarbamoyl bonds in thepeptides M_(b) ³, the comments made above for the proteins Ib apply inanalogous manner.

For the above-described optimisation of the procedure for the chemicalmodification of proteins and for the analysis of the primary structureof such chemically modified proteins, in addition to compounds IA andtheir salts it is also possible to use compounds I wherein R₁, R₂, R₃,R₄ and G are as defined above, R₅ is hydrogen, R₆ is halomethyl and L isan oxygen atom, and salts thereof. These compounds are designatedcompounds IB below.

The comments made above for compounds IA and their salts generally applyin an analogous manner to compounds IB and their salts. However, in thechemical modification of a protein, that is to say in the reaction withthat protein, the compounds IB and their salts react neither with theε-amino groups of lysine building blocks nor with the amino group of theN-terminal amino acid building block of that protein. On the contrary,in the course of the reaction with a compound IB or with a salt thereofspecifically the mercapto groups of cysteine building blocks of aprotein N_(c) of general formula HS-R_(y) (Id), wherein R_(y) in eachcase is the residue of the protein, are converted intocarbonylmethylthio groups, so that a chemically modified protein offormula ##STR4## wherein R₁, R₂, R₃, R₄, R_(y) and G are as definedabove, is obtained. Thus when compounds IB and their salts are used,generally the nature and number of the amino acid building blocksaffected by a chemical modification are likewise clear and,advantageously, narrowly defined, the number of chemically modifiedamino acid building blocks being not greater than the number of cysteinebuilding blocks in the protein under investigation.

The invention therefore relates also to the use of compounds I and theirsalts as adjuncts in the investigation of proteins, for example asreagents for the chemical modification of proteins, especially asreagents for the chemical modification of proteins that is associatedwith coloration. The commercial formulation of the adjuncts may also beincluded.

The present invention relates also to a corresponding process for thechemical modification of proteins, especially a process for the chemicalmodification of proteins that is associated with coloration, whichprocess comprises reacting the proteins with a compound I or with a saltthereof.

The invention relates especially to compounds of formula I wherein R₁ islower alkyl, R₂ is lower alkyl, R₃ is hydrogen, carboxy or sulfo, R₄ iscarboxy or sulfo, G is an unsubstituted or carboxy- and/orsulfo-substituted 1,4-phenylene group or an unsubstituted or carboxy-and/or sulfo-substituted 1,4-naphthylene group and wherein either R₅ andR₆ together are an additional bond and L is an oxygen or sulfur atom, orwherein R₅ is hydrogen, R₆ is halomethyl and L is an oxygen atom, andsalts thereof.

The invention relates especially to compounds of formula I wherein R₁ islower alkyl, R₂ is lower alkyl, R₃ is hydrogen, carboxy or sulfo, R₄ iscarboxy or sulfo, G is an unsubstituted or carboxy- and/orsulfo-substituted 1,4-phenylene group or an unsubstituted or carboxy-and/or sulfo-substituted 1,4-naphthylene group, R₅ and R₆ together arean additional bond and L is an oxygen or sulfur atom, and salts thereof.

The invention relates more especially to compounds of formula I whereinR₁ is C₁ -C₄ alkyl, such as methyl or ethyl, R₂ is C₁ -C₄ alkyl, such asmethyl or ethyl, R₃ is hydrogen or sulfo, R₄ is sulfo, G is anunsubstituted or sulfo-substituted 1,4-phenylene group and whereineither R₅ and R₆ together are an additional bond and L is an oxygen orsulfur atom, or wherein R₅ is hydrogen, R₆ is iodomethyl and L is anoxygen atom, and salts thereof.

The invention relates especially to compounds of formula I wherein R₁ isC₁ -C₄ alkyl, such as methyl or ethyl, R₂ is C₁ -C₄ alkyl, such asmethyl or ethyl, R₃ is hydrogen or sulfo, R₄ is sulfo, G is anunsubstituted or sulfo-substituted 1,4-phenylene group, R₅ and R₆together are an additional bond and L is an oxygen or sulfur atom, andsalts thereof.

The invention relates especially to compounds of formula I wherein R₁ isC₁ -C₄ alkyl, such as methyl, R₂ is C₁ -C₄ alkyl, such as methyl, R₃ ishydrogen, R₄ is sulfo, G is an unsubstituted 1,4-phenylene group andwherein either R₅ and R₆ together are an additional bond and L is asulfur atom, or wherein R₅ is hydrogen, R₆ is iodomethyl and L is anoxygen atom, and salts thereof.

The invention relates more especially to compounds of formula I whereinR₁ is C₁ -C₄ alkyl, such as methyl, R₂ is C₁ -C₄ alkyl, such as methyl,R₃ is hydrogen, R₄ is sulfo, G is an unsubstituted 1,4-phenylene group,R₅ and R₆ together are an additional bond and L is a sulfur atom, andsalts thereof.

The invention relates specifically to the novel compounds of formula Imentioned in the Examples and their salts.

The present invention relates also to a process for the preparation of acompound I or a salt thereof, which process comprises, for example: in acompound of formula ##STR5## or in a salt thereof, converting the NH2group into a group of formula ##STR6## and, if desired, separating amixture of isomers obtainable in accordance with the process into thecomponents and isolating the desired isomer I, resolving a mixture ofenantiomers or diastereoisomers obtainable in accordance with theprocess into the individual enantiomers or diastereoisomers andisolating the desired enantiomer or diastereoisomer, and/or converting afree compound I obtainable in accordance with the process into a salt orconverting a salt obtainable in accordance with the process into thefree compound I or into a different salt.

The reactions described hereinbefore and hereinafter are carried out ina manner known per se, for example in the presence of a catalyst and/orin the absence or, usually, in the presence of a suitable inert solventor diluent or a mixture thereof, the reactions being carried out, asnecessary, with cooling, at room temperature or with heating, forexample in a temperature range of from approximately -80° C. to theboiling temperature of the reaction medium, preferably fromapproximately -20° C. to approximately +150° C., and, if necessary, in aclosed vessel, under pressure, in an inert gas atmosphere and/or underanhydrous conditions.

Some of the starting materials of formulae II, IIa, III, IV, IVa and Vmentioned hereinbefore and hereinafter, which are used in thepreparation of compounds I or their salts, are known or they can beprepared according to methods known per se.

Starting materials having basic centres can, for example, be in the formof acid addition salts, for example with the acids mentioned above,whilst starting compounds having acidic groups can form salts withbases, for example of the kind mentioned above.

The conversion of the NH₂ group in a compound II, or in a salt thereof,into a group Ic can be carried out, for example, by reacting thecompound II or a salt thereof with a compound of formula ##STR7##wherein L is an oxygen or sulfur atom and Z₁ and Z₂ either independentlyof one another each represent a nucleofugal leaving group G₁ or togetherrepresent free or functionally modified oxo G₂ or wherein L is an oxygenatom, Z₁ is a nucleofugal leaving group G₁ and Z₂ is halomethyl.

Nucleofugal leaving groups G₁ are, for example, free, etherified oresterified hydroxy or mercapto groups, also amino, ammonium or sulfoniumgroups. Etherified hydroxy is, for example, lower alkoxy, such asmethoxy or ethoxy, or unsubstituted or substituted phenyl-lower alkoxy,such as unsubstituted or substituted benzyloxy. Esterified hydroxy isespecially hydroxy esterified by a mineral acid or an organic sulfonicacid, especially halogen, such as chlorine, bromine or iodine,sulfonyloxy, such as unsubstituted or halo-substituted loweralkanesulfonyloxy, for example methanesulfonyloxy ortrifluoromethanesulfonyloxy, cycloalkanesulfonyloxy, for examplecyclohexanesulfonyloxy, or unsubstituted or lower alkyl- orhalo-substituted benzenesulfonyloxy, for example benzenesulfonyloxy,p-bromophenylsulfonyloxy or p-toluenesulfonyloxy, also loweralkanoyloxy, for example acetoxy or pivaloyloxy. Etherified mercapto is,for example, lower alkylthio, such as methylthio or ethylthio, orunsubstituted or substituted phenylthio, such as phenylthio orp-tolylthio. Esterified mercapto groups are, for example, loweralkanoylthio groups, such as acetylthio. Amino groups are, for example,amino, N-lower alkylamino, N,N-di-lower alkylamino or N-loweralkanoylamino groups, also N,N-lower alkyleneamino or N,N-aza-, N,N-oxa-or N,N-thia-lower alkyleneamino groups, for example dimethylamino ordiethylamino, also pyrrolidino, piperidino, morpholino orthiomorpholino, also anilino. Ammonium groups are, for example, tertiaryor quaternary ammonium groups corresponding to the amino groupsmentioned above, such as tri-lower alkylammonio or pyridinio. Sulfoniumgroups are, for example, di-lower alkylsulfonium groups, such asdimethylsulfonium.

Free or functionally modified oxo G₂ is, for example, oxo, thioxo or agroup ═N--R'. Groups ═N--R' are, for example, those groups in which R'is hydrogen, lower alkyl or an acyl radical, such as lower alkanoyl,unsubstituted or substituted benzoyl, pyridoyl or lower alkanesulfonyl,for example imino, N-lower alkylimino, N-lower alkanoylimino,unsubstituted or substituted N-benzoylimino or N-loweralkanesulfonylimino groups.

Compounds IIa preferably used for the preparation of compounds I whereinR₅ and R₆ together are an additional bond, or salts thereof, are, forexample, thiophosgene (Z₁ =Z₂ =chlorine) and carbon disulfide (Z₁ and Z₂=thioxo) which result in compounds I wherein L is a sulfur atom, orsalts thereof, and phosgene (Z₁ =Z₂ =chlorine) which results incompounds I wherein L is an oxygen atom, or salts thereof.

Compounds IIa preferably used for the preparation of compounds I whereinR₅ is hydrogen and R₆ is halomethyl, or salts thereof, are, for example,haloacetic acids (Z₁ =hydroxy; Z₂ =halomethyl; L=oxygen atom) andcorresponding reactive haloacetic acid derivatives (Z₁ forexample=halogen, lower alkoxy or sulfonyloxy).

The reaction of a compound II or a salt thereof with a compound IIa iscarried out in customary manner, for example optionally in the presenceof a condensation agent, such as a suitable base, and in the case of thereaction with compounds IIa wherein Z₁ and Z₂ together are thioxo,optionally in the presence of a sulfur-binding agent, and in the case ofthe reaction with compounds IIa wherein Z₁ is G₁, Z₂ is halomethyl and Lis an oxygen atom, optionally in the presence of a water-binding agent,in the absence or, usually, in the presence of a suitable inert solventor diluent or a mixture thereof and, as necessary, with cooling, at roomtemperature or with heating, for example in a temperature range of fromapproximately -80° C. to approximately +200° C., preferably fromapproximately -20° C. to approximately +150° C., and, if necessary, in aclosed vessel, under pressure and/or under an inert gas, such asnitrogen.

Suitable bases are, for example, alkali metal hydroxides, hydrides,amides, alkanolates, carbonates, triphenylmethylides, di-loweralkylamides, amino-lower alkylamides and lower alkylsilylamides, ornaphthaleneamines, lower alkylamines, basic heterocycles, ammoniumhydroxides and carbocyclic amines. Examples that may be mentioned aresodium hydroxide, hydride, amide, ethanolate or carbonate, potassiumtert.butanolate or carbonate, lithium triphenylmethylide, lithiumdiisopropylamide, potassium 3-(aminopropyl)-amide orbis-(trimethylsilyl)amide, or dimethylaminonaphthalene, di- andtri-ethylamine, pyridine, benzyltrimethylammonium hydroxide,1,5-diazabicyclo[4.3.0]non-5-ene (DBN) and1,5-diazabicyclo[5.4.0]undec-5-ene (DBU).

Suitable sulfur-binding agents for the reaction with compounds IIawherein Z₁ and Z₂ together are thioxo are, for example, oxides ofphosphorus, such as tetraphosphorus decaoxide, carbodiimides, such asN,N'-dicyclohexyl carbodiimide, and derivatives of carbonic acid, suchas carbonic acid esters, for example halocarbonic acid lower alkylesters, such as chlorocarbonic acid ethyl esters.

Suitable water-binding agents for the reaction with compounds IIawherein Z₁ is G₁, Z₂ is halomethyl and L is an oxygen atom are, forexample, oxides of phosphorus, such as tetraphosphorus decaoxide, andcarbodiimides, such as N,N'-dicyclohexyl carbodiimide orN-[3-(N,N-dimethylamino)-propyl]-N'-ethyl carbodiimide and the acidaddition salt thereof, for example the hydrochloride.

Inert solvents or diluents that may be mentioned are, for example, waterand, also in the form of mixtures with water, cyclic ethers,unsubstituted or halogenated aromatic hydrocarbons, halogenated loweralkanes, N,N-di-lower alkyl-lower alkanoic acid amides, phosphoricacid-lower alkylamides, di-lower alkylsulfoxides, basic heterocycles andlower alkanols, such as tetrahydrofuran, dioxane, benzene, toluene,xylene, chlorobenzene, 1,2-dichlorobenzene, trichloromethane,N,N-dimethylformamide, hexamethylphosphoric acid triamide, dimethylsulfoxide, pyridine, N-methylmorpholine, methanol and ethanol.

The starting material II or a salt thereof can be prepared analogouslyto known methods, for example by hydrolysis, that is to say by reactionwith water, of a compound of formula ##STR8## wherein Y is an acylradical, or a salt thereof.

Acyl radicals Y are, for example, acyl radicals derived from an organiccarboxylic or sulfonic acid.

Acyl derived from an organic carboxylic acid is, for example, theradical of an aliphatic or monocyclic-aromatic carboxylic acid, such aslower alkanoyl or unsubstituted or substituted benzoyl, also pyridoyl.

Acyl derived from an organic sulfonic acid is, for example, loweralkanesulfonyl.

Lower alkanoyl is, for example, C₂ -C₅ alkanoyl, such as acetyl,propionyl, butyryl, isobutyryl or pivaloyl.

Unsubstituted or substituted benzoyl is, for example, benzoyl,p-chlorobenzoyl or p-nitrobenzoyl.

Lower alkanesulfonyl is, for example, C₁ -C₄ alkanesulfonyl, such asmethane- or ethane-sulfonyl.

The hydrolysis of a compound III or a salt thereof is carried out incustomary manner, for example in the presence of a hydrolysing agent andoptionally in the absence or, usually, in the presence of a suitableinert solvent or diluent or a mixture thereof, the operation beingcarried out, as necessary, with cooling, at room temperature or withheating, for example in a temperature range of from approximately -80°C. to approximately +200° C., preferably from approximately -20° C. toapproximately +150° C., and, if necessary, in a closed vessel, underpressure and/or under an inert gas, such as nitrogen.

Suitable hydrolysing agents are, for example, acids or bases. Suitableacids are, for example, inorganic or organic protonic acids, such asmineral acids, for example sulfuric acid or hydrohalic acids, forexample hydrochloric acid, sulfonic acids, for example loweralkanesulfonic acids or unsubstituted or substituted benzenesulfonicacids, for example methane- or p-toluene-sulfonic acid, or carboxylicacids, for example lower alkanecarboxylic acids, for example aceticacid, whilst bases that can be used are, for example, those mentionedabove, especially sodium or potassium hydroxide.

Suitable inert solvents or diluents are especially those mentionedabove, more especially water and aqueous lower alkanols, such as aqueousmethanol or ethanol.

The starting material III or a salt thereof can be prepared analogouslyto known methods, for example by reaction of a salt of formula ##STR9##wherein A is the anion of a protonic acid, with a compound of formula##STR10## or with a salt thereof.

Anions A of protonic acids in salts IV are, for example, anions of theacids mentioned above for the formation of acid addition salts ofcompounds I, especially anions of strong inorganic protonic acids, suchas anions of mineral acids, for example sulfuric acid, a phosphoric acidor a hydrohalic acid, or of tetrafluoroboric acid, or anions of strongorganic carboxylic acids, such as lower alkanecarboxylic acids, forexample formic acid or acetic acid, for example the sulfate, phosphate,chloride, bromide, tetrafluoroborate or acetate ion.

The reaction of a salt IV with a compound IVa or with a salt thereof iseffected analogously to known procedures under the customary reactionconditions, for example in an inert solvent or diluent, for example ofthe kind mentioned above, preferably in water, optionally in thepresence of an acidic agent, for example in the presence of one of theacids mentioned above, and/or with cooling, at room temperature or withheating, for example in a temperature range of from approximately -20°to approximately +50° C., preferably from approximately 0 toapproximately +30° C.

The starting material IV is known or can be prepared analogously toknown methods, for example by reaction of a compound of formula##STR11## or a salt thereof, with nitrous acid, the reaction beingcarried out under the reaction conditions customarily used, for examplein a solvent or diluent, preferably in water, and/or with cooling, atroom temperature or with heating, for example in a temperature range offrom approximately -20° to approximately +50° C. The nitrous acid ispreferably produced in situ, for example by reaction of an alkali metalnitrite, such as sodium nitrite, with a strong protonic acid, forexample a hydrohalic acid, such as hydrochloric acid, or a loweralkanecarboxylic acid, such as formic acid or glacial acetic acid.

In an especially preferred form, a compound V or a salt thereof isreacted as described above with nitrous acid, which is, for example,formed in situ, and the salt IV initially formed is then furtherreacted, without being isolated and/or additionally purified, in situaccording to the invention with a compound IVa or with a salt thereof toform the desired compound III.

The compounds IVa and their salts are known or can be preparedanalogously to known methods.

The starting material IIa is also known or can be prepared analogouslyto known methods.

Salts of compounds I can be produced in a manner known per se. Thus, forexample, acid addition salts of compounds I are obtained by treatmentwith an acid or a suitable ion exchange reagent. Acidic compounds I canbe converted into salts with bases, for example, by treatment with abase or with a suitable ion exchange reagent. Salts of compounds I canbe converted into the free compounds I in customary manner; for exampleacid addition salts can be converted by treatment with a suitable basicagent, and salts with bases can be converted, for example, by treatmentwith a suitable acidic agent.

Depending upon the procedure and reaction conditions, the compounds Ihaving salt-forming, especially acidic, properties may be obtained infree form or in the form of salts.

As a result of the close relationship between the novel compound I infree form and in the form of its salts, hereinbefore and hereinafter afree compound I or its salts should be understood as meaning also thecorresponding salts or the free compound I, respectively, whereappropriate and expedient.

The novel compounds I, including their salts, can also be obtained inthe form of their hydrates or may include other solvents, for examplethose used for the crystallisation of compounds in solid form.

Depending upon the starting materials and procedures chosen, the novelcompounds I may be in the form of one of the possible isomers or in theform of a mixture thereof. Depending upon the molecular symmetry, forexample depending upon the number and the absolute and relativeconfiguration of the chiral centres, such as asymmetric carbon atoms, aspure isomers there may be obtained, for example, pure enantiomers and/orpure diastereoisomers, such as pure cis/trans isomers or meso-compounds.Accordingly, as isomeric mixtures there may be obtained, for example,enantiomeric mixtures, such as racemates, diastereoisomeric mixtures ormixtures of racemates.

Resulting diastereoisomeric mixtures and mixtures of racemates can beseparated into the pure diastereoisomers or racemates in known manner onthe basis of the physico-chemical differences between the constituents,for example by fractional crystallisation.

Resulting enantiomeric mixtures, such as racemates, can be resolved intothe enantiomers by known methods, for example by recrystallisation froman optically active solvent, by chromatography on chiral adsorbents,with the aid of suitable microorganisms, by cleaving with specific,immobilised enzymes, by means of the formation of inclusion compounds,for example using chiral Crown ethers, in which case only one enantiomeris complexed, or by conversion into diastereoisomeric salts, for exampleby reaction of a basic end product racemate with an optically activeacid, such as a carboxylic acid, for example tartaric or malic acid, ora sulfonic acid, for example camphorsulfonic acid, and separation of themixture of diastereoisomers obtained in this manner, for example on thebasis of their different solubilities, into the diastereoisomers fromwhich the desired enantiomer can be freed by the action of suitableagents. Advantageously, the more active enantiomer is isolated.

The invention also relates to those forms of the preparation processaccording to which a compound obtainable as intermediate at any stage ofthe process is used as starting material and the remaining steps arecarried out, or a starting material is used in the form of a derivativeor salt and/or its racemates or enantiomers or, especially, is formedunder the reaction conditions.

In the preparation process of the present invention it is preferable touse those starting materials which result in the compounds I describedat the beginning as being especially valuable. The invention relatesalso to novel starting materials which were developed specifically forthe preparation of the compounds I, to their use and to processes fortheir preparation, the variables R₁, R₂, R₃, R₄, R₅, R₆, G and L havingthe meanings indicated for the groups of compounds of formula I that arepreferred in each case.

In this connection, special mention should be made of compounds offormula ##STR12## and their salts, to which the comments made above forsalts of compounds I apply in analogous manner. These can be used in anespecially advantageous manner as starting materials for the preparationof compounds I or their salts, for example in accordance with theprocess described above.

The invention accordingly relates also to compounds of formula IIwherein R₁ is lower alkyl, R₂ is lower alkyl, R₃ is hydrogen, carboxy orsulfo, R₄ is carboxy or sulfo, and G is an unsubstituted or substituted1,4-phenylene group or an unsubstituted or substituted 1,4-naphthylenegroup, and salts thereof, to the use of these compounds and their saltsand to a process for the preparation of these compounds and their salts.

The variables in formula II have, for example, the preferred meaningsgiven under formula I.

The invention relates in this respect especially to compounds of formulaII wherein R₁ is lower alkyl, R₂ is lower alkyl, R₃ is hydrogen, carboxyor sulfo, R₄ is carboxy or sulfo, and G is an unsubstituted or carboxy-and/or sulfo-substituted 1,4-phenylene group or an unsubstituted orcarboxy- and/or sulfo-substituted 1,4-naphthylene group, and saltsthereof.

The invention relates in this respect more especially to compounds offormula II wherein R₁ is C₁ -C₄ alkyl, such as methyl or ethyl, R₂ is C₁-C₄ alkyl, such as methyl or ethyl, R₃ is hydrogen or sulfo, R₄ issulfo, and G is an unsubstituted or sulfo-substituted 1,4-phenylenegroup, and salts thereof.

The invention relates in this respect most especially to compounds offormula II wherein R₁ is C₁ -C₄ alkyl, such as methyl, R₂ is C₁ -C₄alkyl, such as methyl, R₃ is hydrogen, R₄ is sulfo, and G is anunsubstituted 1,4-phenylene group, and salts thereof.

The invention relates in this respect specifically to the novelcompounds of formula II mentioned in the Examples and to their salts.

The present invention relates also to a process for the preparation of acompound II or a salt thereof, which process comprises, for example,hydrolysing a compound of formula ##STR13## wherein Y is an acylradical, or a salt thereof, for example as described above, and, ifdesired, separating a mixture of isomers obtainable in accordance withthe process into the components and isolating the desired isomer II,resolving a mixture of enantiomers or diastereoisomers obtainable inaccordance with the process into the individual enantiomers ordiastereoisomers and isolating the desired enantiomer ordiastereoisomer, and/or converting a free compound II obtainable inaccordance with the process into a salt or converting a salt obtainablein accordance with the process into the free compound II or into adifferent salt.

The preparation of compounds III or salts thereof is described above.

Subsequent operations which may, if desired, be carried out on compoundsII or their salts obtainable in accordance with the process or by othermeans are especially separations of enantiomers or diastereoisomers andconversions into one another of salts and free compounds II analogous tothose indicated for compounds I and these operations are also carriedout in analogous manner.

The invention relates also to the use of compounds II or their salts asstarting materials for the preparation of compounds I or their salts.

It is possible, to carry out the above-described optimised procedure forthe chemical modification of proteins and for the analysis of theprimary structure of such chemically modified proteins, that is to saythe sequence consisting of the chemical modification by means of acompound I or a salt thereof, optional denaturation, protease treatmentand HPLC, also automatically, using a device specifically designed forthis purpose.

BRIEF DESCRIPTION OF THE DRAWINGS

The design of the device is schematically shown in FIG. 1.

DETAILED DESCRIPTION

The device comprises a syringe pump (24), which component (24) serves todeliver a sample of the protein which is to be chemically modified bymeans of a compound I or a salt thereof, solvents, reagents and/orcatalysts, that are necessary for the said steps of chemicalmodification by means of a compound I or a salt thereof, optionaldenaturation and protease treatment, inert gas, and washing solutionthrough the connection (1) into the reactor (15), which component (15)is equipped with a screw cap (16) and is installed within aheating-jacket (14), a vacuum pump (25), which component (25) enables bymeans of the connection (2) the evacuation of the reactor (15) for thepurpose of the concentration of the contents of the reactor (15), aconnection (3), which component (3), by means of a slight excesspressure of inert gas delivered from the syringe pump (24) through theconnection (1), serves to deliver the contents of the reactor (15), whenthe chemical modification or the optional denaturation step has beencarried out, through the valve (26), the connection (5), the valve (27)and the connection (9) to the desalting unit (19), or serves to deliverthe contents of the reactor (15), when the protease treatment step hasbeen carried out, through the valve (26), the connection (5), the valve(27) and the connection (7) to the HPLC component (21), to whichcomponent (21) a plotter (22) and through the connection (8) a fractioncollector (20) are attached, or serves to deliver the washing solutioncontained in the reactor (15) through the valve (26) and the connection(11) to the waste reservoir (17), a pump (23), which component (23)delivers eluent through the connection (6), the valve (27) and theconnection (9) to the desalting unit (19), a connection (10), whichcomponent (10) serves to deliver the eluate which is eluted from thedesalting unit (19) to the detector (18), which component (18) serves tocontrol the valve (28) in such a way that the eluate which is elutedfrom the desalting unit (19), when it contains the chemically modifiedprotein prepared in the chemical modification step or optionally thedenatured chemically modified protein prepared in the optionaldenaturation step is delivered through the connection (4) into thereactor (15), and when it contains no such chemically modified proteinor denatured chemically modified protein is delivered through theconnection (12) into the waste reservoir (17), and a connection (13),which component (13) serves to deliver the eluate from the detector (18)to the valve (28). The device according to the invention comprises thecomponents (1) to (28).

The reactor (15) is in its size and shape comparable to a 1.5 mlEppendorf tube. The reactor (15) can be made of any inert material, forexample of glass, virtually inert plastics, such as polytetrafluoroethylene, or high-quality stainless steel, preferably of a transparentmaterial, for example of glass or transparent, virtually inert plastics.The steps of chemical modification, optional denaturation and proteasetreatment are carried out in the taper bottom part of the reactor with atotal reaction volume of from approximately 100 to approximately 300 μl.The reactor can be stoppered by means of the screw cap (16), whichpreferably is made of the same material as the reactor (15). Theconnection (3) should reach as closely as possible to the bottom of thereactor, in order to enable a virtually complete emptying of the reactor(15).

The syringe pump (24) can deliver the educt (the protein which is to bechemically modified), the necessary solvents, reagents, for example thecompound I or a salt thereof used in the chemical modification step,and/or catalysts, as well as inert gas and washing solution into thereactor (15). The syringe pump (24) is a commercially availableapparatus, which is constructed in such a way that kind and quantity ofthe agent which is to be delivered into the reactor (15) can be chosenindependently at any moment. By means of a valve contained in thesyringe pump (24), the connection (1) leading into the reactor (15) canbe stoppered at any time, for example, when the desired quantity ofagent has been pumped into the reactor (15). The quantities which can bepumped in a single step into the reactor (15) can vary within a widevolume range, for example from approximately 1 μl to approximately 200μl.

As vacuum pump (25) any commercially available pump can be used,preferably an oil pump. The desalting unit (19) serves to remove excessreagents and by-products contained in the reaction mixture resultingafter the chemical modification step and the optional denaturation step.Customary chromatographic separation devices can be used as desaltingunit (19), for example devices used for column chromatography, such asmolecular exclusion chromatography, for example gel chromatography. Thedetector (18) is preferably evaluating the light absorption behaviour ofthe molecules, for example the light absorption behaviour at wavelengthsof the ultraviolet or especially of the visible light. Typicalwavelengths are those given hereinbefore for the detector of the HPLCsystem. As heating jacket (14), waste reservoir (17), fraction collector(20), HPLC component (21), plotter (22) and pump (23) which delivers theeluent to the desalting unit (19) suitable commercially availablecomponents can be used.

The valve (26) can open on the one hand the connections (3) and (5) andat the same time stopper the connection (11). On the other hand, valve(26) can open the connections(3) and (11) and at the same time stopperthe connection (5). The valve (27) can either open (5) and (9) and atthe same time stopper (6) and (7), or open (5) and (7) and at the sametime stopper (6) and (9), or open (6) and (9) and at the same timestopper (5) and (7). The valve (28) can either open (4) and (13) and atthe same time stopper (12), or open (12) and (13) and at the same timestopper (4). Commercially available valves of suitable type can be used.

The connections (1) and (3) through (13) can be made of any inertmaterial, for example of glass or virtually inert plastics, such aspolytetrafluoroethylene, preferably of a transparent material, forexample of glass or transparent, virtually inert plastics. Theconnection (2) is made of vacuum resistant tubing.

In detail, the reaction sequence is carried out as follows: A solutionof the protein which is to be chemically modified by a compound I or asalt thereof is delivered by the syringe pump (24) through connection(1) into the reactor (15). Subsequently, a solution of the reagent (acompound I or a salt thereof) is delivered in the same way into thereactor (15). After the chemical modification is finished, a slightexcess pressure of inert gas, for example argon, is built up in thereactor (15) (the inert gas is delivered from the syringe pump (24) viathe connection (1)). The excess pressure causes the contents of thereactor (15) to be delivered via (3), (26), (5), (27) and (9) to thedesalting unit (19). The valves (26) and (27) are adjusted accordingly.The connection (4) is stoppered by means of the valve (28). When thewhole contents of the reactor (15) have been transferred to thedesalting unit (19), the excess pressure is abandoned. The sample isthen passed through the desalting unit (19) in order to purify it, thepump (23) being delivering the eluent via (6), (27) and (9) (the valve(27) is adjusted accordingly). By means of (10), (18), (13), (28) and(4), the purified chemically modified protein is delivered back into thereactor (15), the valve (28) being adjusted accordingly. The detector(18) serves to adjust the valve (28) in such a way, that only thefraction of the eluate containing the desired product is delivered intothe reactor (15), whereas the other fractions are directly delivered viathe connection (12) into the waste reservoir (17). The contents of thereactor (15) (eluate with purified chemically modified protein) areconcentrated by means of the application of vacuum via the connection(2) using the vacuum pump (25). During the evacuation the connection (1)is stoppered by means of the valve contained in the syringe pump. Also,connections (3) and (5), respectively, (by means of the valve (27)) and(4) (by means of the valve (28)) are stoppered.

If a denaturation step is to be performed, then this is done in a manneranalogous to that described for the chemical modification step, usingthe appropriate reagents and solvents. This denaturation step is thenfollowed again by a purification step using the desalting unit (19)analogously. The resulting eluate is again delivered into the reactor(15) and concentrated.

Finally, the protease treatment step is carried out in the reactor (15)in a manner analogous to that described for the chemical modificationstep, again using the appropriate reagents and solvents. When theprotease treatment is finished, the contents of the reactor (15) aredelivered to the HPLC component (21) via (3), (26), (5), (27) and (7)(the valves (26) and (27) are adjusted accordingly) in a manneranalogous to that described before (by means of a slight excess pressureof inert gas). When the HPLC has been performed, the chromatogram isobtained on the plotter (22). The fractions resulting from the HPLC canbe collected by means of the fraction collector (20).

The reaction conditions (temperature, volume, reaction time, and so on)and the gradient of the HPLC are controlled by a pre-set program. Anycatalysts or other adjuncts that are necessary in the describedreactions can be delivered into the reactor (15) by means of the syringepump (24) via (1).

The device is operated in such a fashion, that on the one hand thedesalting unit (19) is continuously washed with eluent delivered fromthe pump (23), while a reaction (chemical modification, optionaldenaturation or protease treatment) is going on in the reactor (15), andthat on the other hand the reactor (15) is cleaned with washing solution(delivered from the syringe pump (24) via (1)), while the sample(chemically modified protein or optionally denatured chemically modifiedprotein) is being purified in the desalting unit (19). The washingsolution of the desalting unit (19) is delivered into the wastereservoir (17) via (10), (18), (13), (28) and (12), and the washingsolution of the reactor (15) is delivered to the waste reservoir (17)via (3), (26) and (11), in each case in a manner analogous to thatdescribed before for the transfer of the products and eluates.

The invention also relates to a device of the type describedhereinbefore specifically designed to carry out automatically thesequence consisting of the chemical modification of a protein by meansof a compound I or a salt thereof, the optional denaturation of thechemically modified protein, the protease treatment of the chemicallymodified and optionally denatured protein, and the HPLC of the peptidemixture resulting from the protease treatment step.

The invention also relates to a process for the manufacture of saiddevice, characterised in that the components of the device describedhereinbefore are combined in the way described hereinbefore to yield thedesired device.

The invention also relates to a process for the automatic chemicalmodification of a protein and for the automatic analysis of the primarystructure of the chemically modified protein consisting of the sequencedescribed hereinbefore, which process is characterised in that theprotein is processed using the device described hereinbefore.

The invention also relates to the use of the device describedhereinbefore for the automatic performance of the sequence describedhereinbefore and to the use of a compound I or a salt thereof as reagentin the chemical modification step performed in said device.

The invention relates also to the following Examples which illustratethe invention described above but are not intended to limit the scopethereof in any way. Temperatures are given in degrees Celsius. HPLCrepresents high-performance liquid chromatography. Lys representslysine. Val represents valine.

EXAMPLE 1

25 ml of 0.24M sodium carbonate solution and 0.91 g (7.9 mmol; 0.6 ml)of thiophosgene are added to 1 g (3.1 mmol) of4-amino-4'-(N,N-dimethylamino)-azobenzene-2-sulfonic acid. The reactionmixture is stirred for 30 minutes at 70°. After cooling to roomtemperature, the resulting product is filtered off by means of a frittedglass filter and thoroughly washed in succession with 1N hydrochloricacid, toluene and again with 1N hydrochloric acid. The black crystalsare then dried in vacuo at room temperature, yielding4'-(N,N-dimethylamino)-4-isothiocyanato-azobenzene-2-sulfonic acid. Theproduct decomposes on heating and has no definite melting point; in theinfra-red spectrum there is a strong band at 2110 cm⁻¹, produced by theasymmetric N═C═S stretch vibration.

4-amino-4'-(N,N-dimethylamino)-azobenzene-2-sulfonic acid can beobtained, for example, as follows:

0.9 g (8.5 mmol) of anhydrous sodium carbonate and 38 ml of water areadded to 3.5 g (15 mmol) of 5-(N-acetylamino)-2-aminobenzenesulfonicacid. The reaction mixture is stirred until a clear solution has beenobtained. This solution is then cooled to +10° by means of an ice bath.After the addition of 1.22 g (17.7 mmol) of solid sodium nitrite themixture is stirred for a further 2 minutes. The reaction mixture is thenpoured onto a mixture of 3.5 ml of concentrated hydrochloric acid and 25g of crushed ice. Stirring is continued for a further 30 minutes at 0°and then a solution of 2.01 g (16.6 mmol; 2.1 ml) of N,N-dimethylanilinein 3 ml of glacial acetic acid is added to the reaction mixture. Thetemperature of the reaction mixture is allowed to rise to roomtemperature over a period of 3 to 4 hours. The precipitated product isfiltered off and dried overnight in vacuo at room temperature, yielding4-(N-acetylamino)-4'-(N,N-dimethylamino)-azobenzene-2-sulfonic acid inthe form of black to purple crystals which decompose on heating and haveno definite melting point.

2 g (5.5 mmol) of4-(N-acetylamino)-4'-(N,N-dimethylamino)-azobenzene-2-sulfonic acid aredissolved in 8 ml of ethanol. After the addition of 4 ml of 11N sodiumhydroxide solution, the mixture is stirred for 60 minutes at 90°. Themixture is then allowed to cool to room temperature and then 5 ml of11.5N hydrochloric acid are added. The reaction mixture is then leftovernight at +4°. The precipitated product is filtered off and washedthoroughly with 1N hydrochloric acid. After drying in vacuo at roomtemperature, 4-amino-4'-(N,N-dimethylamino)-azobenzene-2-sulfonic acidis obtained in the form of black to purple crystals which decompose onheating and have no definite melting point.

EXAMPLE 2

0.67 g (2.1 mmol) of4-amino-4'-(N,N-dimethylamino)-azobenzene-2-sulfonic acid (Example 1)are dissolved in 5 ml of absolute pyridine. This solution is then addeddropwise, with stirring and while cooling with ice/sodium chloride, to amixture of 424 mg (2.1 mmol) of N,N'-dicyclohexyl carbodiimide, 5 ml ofanhydrous pyridine and 1.26 g (16.6 mmol; 1 ml) of carbon disulfide. Thereaction mixture is then stirred for 3 to 4 hours with the cooling beingmaintained. The mixture is then left to stand at room temperature for 17hours. The pyridine and excess carbon disulfide are then removed ascompletely as possible under reduced pressure. Column chromatography ofthe residue on silica gel (150 to 200 mesh) with benzene as eluantyields 4'-(N,N-dimethylamino)-4-isothiocyanato-azobenzene-2-sulfonicacid which crystallises out when the eluate is concentrated byevaporation.

EXAMPLE 3

0.67 g (2.1 mmol) of4-amino-4'-(N,N-dimethylamino)-azobenzene-2-sulfonic acid (Example 1)are dissolved in 5 ml of absolute pyridine. With stirring and whilecooling with ice, 1.26 g (16.6 mmol; 1 ml) of carbon disulfide and 0.22g (2.2 mmol; 0.3 ml) of triethylamine are added to the solution. Thereaction mixture is then stirred for 3 to 4 hours with the cooling beingmaintained. The mixture is then left to stand for 17 hours at roomtemperature and is then concentrated to dryness by evaporation. Thetriethylammonium dithiocarbamate remaining behind as residue is driedovernight in vacuo and is then dissolved in 12 ml of trichloromethane.While cooling with ice, 0.22 g (2.2 mmol; 0.3 ml) of triethylamine isadded to the solution. To this mixture there is then added dropwise,within a period of 4 minutes, 0.34 g (3.1 mmol; 0.3 ml) ofchlorocarbonic acid ethyl ester, with stirring and while cooling withice. The reaction mixture is left to stand for 3 to 4 hours with thecooling being maintained and is then concentrated to dryness byevaporation under reduced pressure. The crude product remaining behindas residue is purified by column chromatography as described in Example2, yielding4'-(N,N-dimethylamino)-4-isothiocyanato-azobenzene-2-sulfonic acid.

EXAMPLE 4

In a manner analogous to that described in Examples 1 to 3, startingfrom 4-amino-4'-(N,N-dimethylamino)-azobenzene-2,6-disulfonic acid and4-(N,N-dimethylamino)-naphthalene-1-azo-(4'-aminobenzene-2'-sulfonicacid), it is also possible to obtain4'-(N,N-dimethylamino)-4-isothiocyanato-azobenzene-2,6-disulfonic acidand4-(N,N-dimethylamino)-naphthalene-1-azo-(4'-isothiocyanatobenzene-2'-sulfonicacid), respectively.

The starting materials can each be obtained in a manner analogous tothat described in Example 1, starting from4-(N-acetylamino)-4'-(N,N-di-methylamino)-azobenzene-2,6-disulfonic acidand4-(N,N-dimethylamino)naphthalene-1-azo-[4'-(N-acetylamino)-benzene-2'-sulfonicacid], respectively.

EXAMPLE 5

20 ml of chlorobenzene are cooled to 0° by cooling with ice/sodiumchloride. 1.1 g (11.1 mmol) of phosgene are introduced by condensationinto the cooled chlorobenzene with the customary precautions beingobserved. With the cooling being maintained and with vigorous stirring,a solution of 1.79 g (5.6 mmol) of4-amino-4'-(N,N-dimethylamino)-azobenzene-2-sulfonic acid (Example 1) in30 ml of chlorobenzene is added dropwise at such a rate that thetemperature of the reaction mixture does not exceed +25°. When thedropwise addition is complete, the cooling bath is replaced by a heatingbath by means of which the reaction mixture is heated to 130° within aperiod of 90 minutes while constant stirring is continued. In the courseof this process, as soon as the reaction mixture has reached atemperature of 75°, further phosgene is slowly introduced until a clearsolution is obtained. The reaction mixture is then heated under reflux,with nitrogen being passed through the mixture, until phosgene can nolonger be detected in the outgoing stream of gas (about 2 hours). Aftercooling to room temperature, the reaction mixture is concentrated ascompletely as possible under reduced pressure. Column chromatography ofthe residue on silica gel (150 to 200 mesh) with benzene as eluantyields 4'-(N,N-dimethylamino)-4-isocyanato-azobenzene-2-sulfonic acidwhich crystallises out when the eluate is concentrated by evaporation.

EXAMPLE 6

In a manner analogous to that described in Example 5, starting from4-amino-4'-(N,N-dimethylamino)-azobenzene-2,6-disulfonic acid and4-(N,N-dimethylamino)-naphthalene-1-azo-(4'-aminobenzene-2'-sulfonicacid) it is also possible to obtain4'-(N,N-dimethylamino)-4-isocyanato-azobenzene-2,6-disulfonic acid and4-(N,N-dimethylamino)-naphthalene-1-azo-(4'-isocyanatobenzene-2'-sulfonicacid), respectively.

The starting materials can each be obtained in a manner analogous tothat described in Example 4.

EXAMPLE 7

1.8 g (9.7 mmol) of iodoacetic acid and 1.2 g (6.3 mmol) ofN-[3-(N,N-dimethylamino)-propyl]-N'-ethyl-carbodiimide hydrochloride aredissolved in 9 ml of water. This solution is immediately mixed, withstirring, with a solution of 0.5 g (1.6 mmol) of4-amino-4'-(N,N-dimethylamino)-azobenzene-2-sulfonic acid (Example 1) in7.6 ml of a buffer solution which has a pH of 9 and consists of 50 mMsodium hydrogen carbonate solution and 50 mM sodium carbonate solution.0.4 ml of 11N sodium hydroxide solution is then added dropwise to themixture which is then stirred for 10 minutes at room temperature. 1Nhydrochloric acid is then added dropwise until the mixture turns purple.The product that precipitates is filtered off, washed thoroughly with100 ml of 0.1N hydrochloric acid and dried in vacuo. The dried crudeproduct is dissolved in a mixture of 16 ml of N,N-dimethylformamide and0.4 ml of triethylamine. Further precipitation with 1N hydrochloric acidin the manner described above, filtration and drying of the filtercontents in vacuo yield the desired pure4'-(N,N-dimethylamino)-4-(N-iodoacetyl)-amino-azobenzene-2-sulfonic acidwhich decomposes on heating and has no definite melting point.

EXAMPLE 8

In a manner analogous to that described in Example 7, starting from4-amino-4'-(N,N-dimethylamino)-azobenzene-2,6-disulfonic acid and4-(N,N-dimethylamino)-naphthalene-1-azo-(4'-aminobenzene-2'-sulfonicacid), it is also possible to obtain4'-(N,N-dimethylamino)-4-(N-iodoacetyl)-amino-azobenzene-2,6-disulfonicacid and4-(N,N-dimethylamino)-naphthalene-1-azo-[4'-(N-iodoacetyl)-aminobenzene-2'-sulfonicacid], respectively.

The starting materials can each be obtained in a manner analogous tothat described in Example 4.

EXAMPLE 9

Monoclonal antibodies that act specifically against eglin C arechemically modified at room temperature over a period of 18 hours in a50 mM sodium hydrogen carbonate solution by the action of a 2 mMsolution of4'-(N,N-dimethylamino)-4-isothiocyanato-azobenzene-2-sulfonic acid.Subsequent structural analysis shows that each antibody molecule hasbeen chemically modified by 30 molecules of4'-(N,N-dimethylamino)-4-isothiocyanato-azobenzene-2-sulfonic acid. Withthis degree of modification, the antibodies exhibit specificity againstand affinity for recombinant eglin C that is unchanged in comparisonwith the unmodified antibodies. These chemically modified antibodieswhich have also been coloured at the same time can be used todemonstrate the presence of eglin C with the aid of the sandwich dotassay. In that assay concentrations of 1 μg/ml and above can be detectedwith the naked eye.

EXAMPLE 10

Hirudin, a specific inhibitor of thrombin, is chemically modified at 37°over a period of 7 hours in a 50 mM sodium hydrogen carbonate solutionby the action of a 2 mM solution of4'-(N,N-dimethylamino)-4-isothiocyanato-azobenzene-2-sulfonic acid. Thehirudin so modified is subjected to gel chromatography on a Sephadex G25 column. In the modification of the inhibitor, 1.96 mol of4'-(N,N-dimethylamino)-4-isothiocyanato-azobenzene-2-sulfonic acid arereacted per mol of hirudin. The structure of the modified inhibitor andits biological activity are analysed and the following findings areobtained:

a) The chemically modified hirudin has a reduced inhibitor activityagainst thrombin, as can be seen from the fact that the correspondingdissociation constant K_(i) is 300 times greater.

b) Monoclonal antibodies against native hirudin act also against thechemically modified hirudin.

c) The chemically modified hirudin is carboxymethylated and treated withprotease V8. The resulting peptides are subjected to HPLC. At awave-length of 536 nm two peptides are detected. Structural analysisshows that these two coloured peptides correspond to amino acid buildingblocks 1 to 7 and 18 to 37 and that selectively the building blocksVal-1 and Lys-27 are modified by4'-(N,N-dimethylamino)-4-isothiocyanato-azobenzene-2-sulfonic acid.

EXAMPLE 11

Human antithrombin is chemically modified at room temperature over aperiod of 15 minutes in a 50 mM sodium hydrogen carbonate solution bythe action of a 1 mM solution of4'-(N,N-dimethylamino)-4-isothiocyanato-azobenzene-2-sulfonic acid. Theantithrombin so modified is subjected to gel chromatography on aSephadex G 25 column. The structure of the modified antithrombin and itsbiological activity are analysed and the following findings areobtained:

a) The chemically modified antithrombin retains only 25% of its heparincofactor activity, but its inhibitor activity against thrombin in theabsence of heparin (progressive inhibitor activity) is virtuallyunchanged.

b) The chemically modified antithrombin is carboxymethylated and treatedwith trypsin. The resulting peptides are subjected to HPLC. At awave-length of 436 nm three peptides are detected. Structural analysisshows that these three coloured peptides correspond to amino acidbuilding blocks 91 to 111, 115 to 129 and 133 to 139 and thatselectively the building blocks Lys-107, Lys-125 and Lys-136 aremodified by4'-(N,N-dimethylamino)-4-isothiocyanato-azobenzene-2-sulfonic acid.

c) If antithrombin is incubated with heparin before being acted upon by4'-(N,N-dimethylamino)-4-isothiocyanato-azobenzene-2-sulfonic acid, thechemical modification of the building blocks Lys-107, Lys-125 andLys-136 is inhibited by 98%, 88% and 93%, respectively.

d) The building blocks Lys-107, Lys-125 and Lys-136 of humanantithrombin are accordingly of particular importance to the bonding ofheparin.

EXAMPLE 12

In a manner analogous to that described in Examples 9 to 11 it is alsopossible to use a different compound of formula I or a salt of acompound of formula I, for example according to Examples 1 to 8, for thechemical modification of a protein.

EXAMPLE 13

The investigation of the heparin binding site of human antithrombin bymeans of a device of the type shown in FIG. 1 can be carried out asfollows:

a) Human antithrombin (432 amino acids) and the heparin-antithrombincomplex are processed one after another, the following steps b) throughg) being carried out in each case. The device used in these stepscomprises the components shown schematically in FIG. 1. The reactor (15)is made of glass. The detector (18) works on the basis of the absorptionof light of a wavelength of 436 nm. The connections (1) and (3) through(13) are made of glass. The connection (2) is made of vacuum resistanttubing. An oil pump is used as vacuum pump (25). The desalting unit (19)consists of a Sephadex G 25 column.

b) Chemical modification step

Solvent: 50M sodium hydrogencarbonate solution (pH=8.3);

Sample 1: antithrombin, 250 μg;

Sample 2: heparin-antithrombin complex (2:1 parts by weight), 750 μg(250 μg of antithrombin and 500 μg of heparin);

Reagent of the formula I for the chemical modification: 1 mM solution of4'-(N,N-dimethylamino)-4-isothiocyanato-azobenzene-2-sulfonic acid inthe solvent mentioned;

Reaction volume: 200 μl;

Reaction temperature: +25°;

Reaction time: 7.5 minutes.

c) Desalting step

Column: Sephadex G 25 (length: 3.5 cm; internal diameter: 1 cm);

Eluent: 50 mM ammonium hydrogen carbonate solution (pH=8.0).

d) Denaturation Step

Solvent: 0.5M α,α,α-tris(hydroxymethyl)methylaminehydrochloride ("trishydrochloride") solution (pH=8.4) and 5M guanidine hydrochloridesolution;

Reaction volume: 200 μl;

Reduction reagent: threo-1,4-dimercapto-2,3-butanediol (dithiothreitol,DTT), 1 mg;

Reduction temperature: +37°;

Reduction time: 2 hours;

Carboxymethylation reagent: iodoacetic acid, 2 mg;

Carboxymethylation temperature: +37°;

Carboxymethylation time: 15 minutes.

e) Desalting step

Column: Sephadex G 25 (length: 3.5 cm; internal diameter: 1 cm); Eluent:50 mM ammonium hydrogen carbonate solution (pH=8.0).

f) Protease treatment step

Protease: trypsin, 10 μg;

Solvent: 50 mM ammonium hydrogen carbonate solution (pH=8.0);

Reaction volume: 100 μl;

Reaction temperature: +37°;

Reaction time: 2 hours.

g) HPLC step

Column: Vydac C-18 for peptides and proteins;

Column temperature: +25°;

Solvent A: 17.5 mM sodium acetate (pH=5.0);

Solvent B: acetonitrile;

Gradient: linear from 10% B to 70% B within 30 minutes;

Flow rate: 1 ml per minute;

Detector: absorption of light of a wavelength of 436 nm.

h) The comparison of the peak patterns in the two chromatogramsresulting from sample 1 and sample 2, respectively, and the sequenceanalysis of the coloured peptides which correspond to peaks that appearonly in the chromatogram of sample 1 reveal, that the amino acidbuilding blocks Lys-107, Lys-125 and Lys-136 are situated within theheparin binding site of human antithrombin.

EXAMPLE 14

In a manner analogous to that described in Example 13, the investigationof the hirudin binding site of human thrombin can be carried out usingthe device described in Example 13. Human thrombin (sample 1; 250 μg)and the hirudin-thrombin complex [sample 2; 750 μg; molar ratio(hirudin: thrombin)=(1,2:1)] are processed one after another, steps b)through g) of Example 13 being carried out analogously in each case. Thecomparison of the peak patterns in the two chromatograms resulting fromsample 1 and sample 2, respectively, and the sequence analysis of thecoloured peptides which correspond to peaks that appear only in thechromatogram of sample 1 reveal, that the amino acid building blocksLys-21, Lys-52, Lys-65, Lys-106, Lys-107 and Lys-154 are situated withinthe hirudin binding site of human thrombin.

EXAMPLE 15

In a manner analogous to that described in Examples 13 and 14 it is alsopossible to use a different compound of formula I or a salt of acompound of formula I, for example according to Examples 1 to 8, asreagent for the chemical modification in step b) of the processingsequence carried out in the device described in Example 13.

What is claimed is:
 1. A process for the chemical modification of aprotein comprising the conversion of an amino group in the ε-position ofa lysine building block of the protein to a carbamoyl or athiocarbamoyl, or the conversion of a mercapto group of a cysteinebuilding block of the protein into a carbonylmethylthio group, whereinthe protein is reacted with a compound of formula I ##STR14## wherein R₁is lower alkyl;R₂ is lower alkyl; lower is 1-7 carbon atoms; R₃ ishydrogen, carboxy, or sulfo; R₄ is carboxy or sulfo; G is a1,4-phenylene group which is unsubstituted or is substituted by 1-4substituents selected from carboxy and sulfo or G is a 1,4-naphthylenegroup which is unsubstituted or substituted by 1-6 substituents selectedfrom carboxy and sulfo; and wherein R₅ and R₆ together are an additionalbond; and L is an oxygen atom or a sulfur atom; or wherein R₅ ishydrogen; R₆ is halomethyl; and L is an oxygen atom,or a salt thereof.2. A process for the chemical modification of a protein that isassociated with coloration according to claim 1, wherein the procedureis as indicated in claim
 1. 3. A process according to claim 1 for thechemical modification of lysine building blocks of a protein, whereinthe protein is reacted with4'-(N,N-dimethylamino)-4-isothiocyanato-azobenzene-2-sulfonic acid.
 4. Aprocess according to claim 1 for the chemical modification of cysteinebuilding blocks of a protein, wherein the protein is reacted with4'-(N,N-dimethylamino)-4-(N-iodoacetyl)-amino-azobenzene-2-sulfonicacid.
 5. The process of claim 1 wherein the compound of formula I is4'-(N,N-dimethylamino)-4-isothiocyanato-azobenzene-2-sulfonic acid. 6.The process of claim 1 wherein the compound of formula I is4'-(N,N-dimethylamino)-4-(N-iodoacetyl)-amino-azobenzene-2-sulfonicacid.